Utjecaj epigenetički aktivnih tvari na rast i razvoj eksperimentalnog tumora zametnih stanica miša in vitro

doctoral thesis

• Title (english): Impact of epigenetic agents on growth and development of experimental mouse germ cell tumour in vitro
• Author: Robert Buljubašić
• Mentor: Nino Sinčić (mentor)
• Committee member: Vlatka Zoldoš (predsjednik povjerenstva)
• Committee member: Jerka Dumić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2020-09-09, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology
• Universal decimal classification (UDC): 57 - Biological sciences. Physical anthropology. Biodiversity

Abstract

Tumori zametnih stanica (TZS) testisa najčešća su neplastična bolest mlađe muške populacije s trendom porasta incidencije u Republici Hrvatskoj. Čini se da su inicijacija i progresija TZS pod izravnim utjecajem okoliša koji često rezultiraju epigenetičkim promjenama. Epigenetičke promjene su reverzibilne, a mogu biti jatrogeno modulirane epigenetički aktivnim lijekovima. Cilj disertacije bio je proučiti učinak epigenetički aktivnih molekula iz grupe inhibitora DNA metilaza, histonskih deacetilaza i inhibitora RNA interferencijskih molekula kao što su redom 5-azacitidin, trihostatin A, valproat, esiOct3/4, esiNanog i esiTrrap na rast i razvoj eksperimentalnog TZS-a miša u posebnom in vitro modelu. Eksperimentalni TZS in vitro razvijeni su kultiviranjem gastrulirajućeg zametnog cilindra. Zametni cilindri bili su jednokratno tretirani dva sata po izolaciji te kultivirani u standardnim uvjetima na granici tekućine i zraka tijekom sedam dana. Tumorska su tkiva analizirana metodama otiska po Westernu, imunohistokemije, digital droplet PCR i pirosekvenciranja nakon bisulfitne konverzije DNA na biljege razvojnih procesa. Inhibitori 5-azacitidin, trihostatin A, valproat i esiOct3/4 izazvali su drastično smanjenje rasta i razvoja tumora potičući apoptozu, smanjujući populaciju matičnih stanica te inducirajući diferencijaciju. Zaključno, ovo istraživanje dokazalo je da epigenetički aktivne molekule utječu na proces tumorigeneze djelujući na ishodišne normalne stanice, ali da imaju i terapijski potencijal za primjenu u liječenju TZS.

Abstract (english)

Germ cell tumours (GCT) of testis are most common neoplasias in young male population with increasing incidence in Republic of Croatia. It seems that GCT initiation and progression directly depend on the environment and epigenetic alterations. Epigenetic alterations are reversible and can be iatrogenically modulated by epigenetically active drugs. The aim of this dissertation was to study epigenetically active molecules from the groups of DNA methyltransferase inhibitors, HDAC inhibitors and RNA interfering molecules such as 5-azacytidine, Trichostatin-A, Valproate, esiOct3/4, esiNanog, and esiTrrap, respectively, on the growth and development of experimental mouse GCT in a specific in vitro model where GCTs developed from the gastrulating mouse embryo cylinder. The embryo cylinders were subjected to a single treatment for two hours after isolation, and were cultivated in standard conditions at the air-liquid interface for seven days. Tumours were analysed by Western blot, immunohistochemistry, digital droplet PCR, and pyrosequencing. 5-azacytidine, Trichostatin-A, Valproate and esiOct3/4 caused drastic reduction in tumour growth and development by promoting apoptosis, reducing stem cell populations, and inducing differentiation. In conclusion, this study proved that epigenetically active substances influence the process of tumorigenesis though their action on the original source of the tumour, the normal embryo, and have the potential for use in the GCT therapy.

• Keywords: Tumori zametnih stanica; epigenetički inhibitori; 5-azacitidin; trihostatin-A; valproat; esiRNA
• Keywords (english): germ cell tumours; epigenetic inhibitors; 5-azacytidine; Trichostatin-A; Valproate; esiRNA
• Language: croatian
• URN:NBN: urn:nbn:hr:217:675814
• Promotion: 2021
• Study programme: Title: Biology; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija (doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-01-29 12:37:22